Przywara D A, Kulkarni J S, Wakade T D, Leontiev D V, Wakade A R
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
J Neurochem. 1998 Nov;71(5):1889-97. doi: 10.1046/j.1471-4159.1998.71051889.x.
Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24-48 h. Uptake of [3H]norepinephrine ([3H]NE) was used as an index of survival and decreased >50% in NGF-deprived cultures within 24 h. PACAP (1-100 nM) restored [3H]NE uptake to 92 +/- 8% of that of NGF-supported controls. Depolarization-induced [3H]NE release in neurons rescued by PACAP was the same as that in NGF-supported neurons. PACAP rescue was not mimicked by forskolin or 8-bromo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis-associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 +/- 40 versus 38 +/- 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF-deprived, PACAP-rescued cultures (14 +/- 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF-deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.
从交感神经元中去除神经生长因子（NGF）会启动神经元死亡程序并导致细胞凋亡。我们发现垂体腺苷酸环化酶激活多肽（PACAP）可防止NGF缺乏的交感神经元发生凋亡。在接种时添加到培养基中的PACAP（100 nM）无法支持神经元存活。然而，在含有NGF培养2天然后去除NGF的神经元中，PACAP在接下来的24 - 48小时内可防止细胞死亡。[3H]去甲肾上腺素（[3H]NE）的摄取被用作存活指标，在去除NGF的培养物中，24小时内[3H]NE摄取减少超过50%。PACAP（1 - 100 nM）可将[3H]NE摄取恢复至NGF支持的对照水平的92±8%。由PACAP挽救的神经元中，去极化诱导的[3H]NE释放与NGF支持的神经元中的相同。福斯可林或8 - 溴环磷酸腺苷不能模拟PACAP的挽救作用，蛋白激酶A抑制剂Rp - 腺苷3',5' - 环磷酸硫代酯也不能阻断其作用。毒蕈碱介导的磷脂酰肌醇动员不能支持NGF缺乏的神经元。因此，PACAP可能利用新的信号传导途径来促进交感神经元的存活。在去除NGF的6小时内，与细胞凋亡相关的半胱天冬酶CPP32活性增加约四倍（145±40对38±17 nmol底物裂解/分钟/毫克蛋白质），在NGF缺乏但经PACAP挽救的培养物中甚至恢复到低于对照水平（14±7 nmol/分钟/毫克蛋白质）。向NGF缺乏的培养物中重新添加NGF或PACAP可逆转CPP32的激活，而这被20S蛋白酶体的强效特异性抑制剂乳胞素所阻断，这表明NGF和PACAP将CPP32靶向蛋白酶体进行降解。由于PACAP是自主神经节中的节前神经递质，我们提出当NGF供应受损时，这种神经递质作为交感神经元凋亡挽救者的新功能。
